Shorter Alkanesulfonate Carbon Chains Destabilize the Active Site Architecture of SsuD for Desulfonation.

Bacteria have evolved to utilize alternative organosulfur sources when sulfur is limiting. The SsuE/SsuD and MsuE/MsuD enzymes expressed when sulfur sources are restricted, are responsible for providing specific bacteria with sulfur in the form of alkanesulfonates. In this study, we evaluated why two structurally and functionally similar FMNH2-dependent monooxygenase enzymes (MsuD and SsuD) are needed for the acquisition of alkanesulfonates in some bacteria. In desulfonation assays, MsuD was able to utilize the entire range of alkanesulfonates (C1-C10). However, SsuD was not able to utilize smaller alkanesulfonate substrates. Interestingly, SsuD had a similar binding affinity for methanesulfonate (MES) (15 ± 1 µM) as MsuD (12 ± 1 µM) even though SsuD was not able to catalyze the desulfonation of the MES substrate. SsuD and MsuD showed decreased proteolytic susceptibility in the presence of FMNH2 with MES and octanesulfonate (OCS). Tighter loop closure was observed for the MsuD/FMNH2 complex with MES and OCS compared to SsuD under comparable conditions. Analysis of the SsuD/FMNH2/MES structure using accelerated molecular dynamics simulations found three different conformations for MES, demonstrating the instability of the bound structure. Even when MES was bound in a similar fashion to OCS within the active site, the smaller alkane chain resulted in a shift of FMNH2 so that it was no longer in a position to catalyze the desulfonation of MES. The active site of SsuD requires a longer alkane chain to maintain the appropriate architecture for desulfonation.

Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1).

Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences.

Hydration of divalent lanthanides, Sm2+ and Eu2+ : A molecular dynamics study with polarizable AMOEBA force field.

The chemistry of divalent lanthanides, Ln2+ , is a growing sub-field of heavy element chemistry owing to new synthetic approaches. However, some theoretical aspects of these unusual cations are currently underdeveloped, especially as they relate to their dynamic properties in solution. In this work, we address the hydration of two of the classical Ln2+ cations, Sm2+ and Eu2+ , using atomic multipole optimized energetic for biomolecular applications (AMOEBA) force fields. These cations have not been parameterized to date with AMOEBA, and few studies are available because of their instability with respect to oxidation in aqueous media. Coordination numbers (CN's) of 8.2 and 8.1 respectively for Sm2+ and Eu2+ , and 8.8 for both Sm3+ and Eu3+ have been obtained and are in good agreement with the few available AIMD and X-ray absorption fine structures studies. The decreased CN of Ln2+ compared with Ln3+ arises from progressive water exchange events that indicates the gradual stabilization of 8-coordinate structures with respect to 9-coordinate geometries. Moreover, the effects of the chloride counter anions on the coordination of Ln2+ cations have been studied at different chloride concentrations in this work. Lastly, water exchange times of Ln2+ cations have been calculated to provide a comprehensive understanding of the behavior of Eu2+ and Sm2+ in aqueous chloride media.

Substrate-Dependent Mobile Loop Conformational Changes in Alkanesulfonate Monooxygenase from Accelerated Molecular Dynamics.

Substrate-induced conformational changes present in alkanesulfonate monooxygenase (SsuD) are crucial to catalysis and lead to distinct interactions between a dynamic loop region and the active site. Accelerated molecular dynamics (aMD) simulations have been carried out to examine this potential correlation by studying wild-type SsuD and variant enzymes bound with different combinations of reduced flavin (FMNH2), C4a-peroxyflavin intermediate (FMNOO-), and octanesulfonate (OCS). Three distinct mobile loop conformations were identified: "open", "closed", and "semiclosed". The substrate-free SsuD system possessed a wide opening capable of providing full access for substrates to enter the active site. Upon binding FMNH2, SsuD adopts a closed conformation that would prevent unproductive oxidation reactions in the absence of OCS. Two salt bridges, Asp111-Arg263 and Glu205-Arg271, were identified as particularly important in maintaining the closed conformation. Experimental substitution of Arg271 to Ala did not alter the catalytic activity, but the variant in the presence of reduced flavin was more susceptible to proteolytic digestion compared to wild-type. With both FMNH2 and OCS bound in SsuD, a second conformation was formed dependent upon a favorable π-π interaction between His124 and Phe261. Accordingly, there was no observed activity with the F261W SsuD variant in steady-state kinetic assays. This semiclosed conformation may be more appropriate for accepting O2 into the binding pocket and/or may properly orient the active site for the ensuing oxygenolytic cleavage. Finally, simulations of SsuD simultaneously bound with FMNOO- and OCS found an open mobile loop region that suggests alternative flavin intermediates may participate in the reaction mechanism.

Accurate Diels-Alder Energies and Endo Selectivity in Ionic Liquids Using the OPLS-VSIL Force Field.

Our recently developed optimized potentials for liquid simulations-virtual site ionic liquid (OPLS-VSIL) force field has been shown to provide accurate bulk phase properties and local ion-ion interactions for a wide variety of imidazolium-based ionic liquids. The force field features a virtual site that offloads negative charge to inside the plane of the ring with careful attention given to hydrogen bonding interactions. In this study, the Diels-Alder reaction between cyclopentadiene and methyl acrylate was computationally investigated in the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [BMIM][PF6], as a basis for the validation of the OPLS-VSIL to properly reproduce a reaction medium environment. Mixed ab initio quantum mechanics and molecular mechanics (QM/MM) calculations coupled to free energy perturbation and Monte Carlo sampling (FEP/MC) that utilized M06-2X/6-31G(d) and OPLS-VSIL gave activation free energy barriers of 14.9 and 16.0 kcal/mol for the endo-cis and exo-cis Diels-Alder reaction pathways, respectively (exptl. ΔH‡ of 14.6 kcal/mol). The endo selectivity trend was correctly predicted with a calculated 73% endo preference. The rate and selectivity enhancements present in the endo conformation were found to arise from preferential hydrogen bonding with the exposed C4 ring hydrogen on the BMIM cation. Weaker electronic stabilization of the exo transition state was predicted. For comparison, our earlier ±0.8 charge-scaled OPLS-2009IL force field also yielded a ΔG‡ of 14.9 kcal/mol for the favorable endo reaction pathway but did not adequately capture the highly organized solvent interactions present between the cation and Diels-Alder transition state.

Examining Product Specificity in Protein Arginine Methyltransferase 7 (PRMT7) Using Quantum and Molecular Mechanical Simulations.

Protein arginine methyltransferase 7 (PRMT7) catalyzes the formation of monomethylarginine (MMA) but is incapable of performing a dimethylation. Given that PRMT7 performs vital functions in mammalian cells and has been implicated in a variety of diseases, including breast cancer and age-related obesity, elucidating the origin of its strict monomethylation activity is of considerable interest. Three active site residues, Glu172, Phe71, and Gln329, have been reported as particularly important for product specificity and enzymatic activity. To better understand their roles, mixed quantum and molecular mechanical (QM/MM) calculations coupled to molecular dynamics and free energy perturbation theory were carried out for the WT, F71I, and Q329S trypanosomal PRMT7 (TbPRMT7) enzymes bound with S-adenosyl- L-methionine (AdoMet) and an arginine substrate in an unmethylated or methylated form. The Q329S mutation, which experimentally abolished enzymatic activity, was appropriately computed to give an outsized Δ G‡ of 30.1 kcal/mol for MMA formation compared to 16.9 kcal/mol for WT. The F71I mutation, which has been experimentally shown to convert the enzyme from a type III PRMT into a mixed type I/II capable of forming dimethylated arginine products, yielded a reasonable Δ G‡ of 21.9 kcal/mol for the second turnover compared to 28.8 kcal/mol in the WT enzyme. Similar active site orientations for both WT and F71I TbPRMT7 allowed Glu172 and Gln329 to better orient the substrate for SN2 methylation, enhanced the nucleophilicity of the attacking guanidino group by reducing positive charge, and facilitated the binding of the subsequent methylated products.

Phe71 in Type III Trypanosomal Protein Arginine Methyltransferase 7 (TbPRMT7) Restricts the Enzyme to Monomethylation.

Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal SN2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.

Salt stress response triggers activation of the jasmonate signaling pathway leading to inhibition of cell elongation in Arabidopsis primary root.

Salinity is a severe abiotic stress that affects irrigated croplands. Jasmonate (JA) is an essential hormone involved in plant defense against herbivory and in responses to abiotic stress. However, the relationship between the salt stress response and the JA pathway in Arabidopsis thaliana is not well understood at molecular and cellular levels. In this work we investigated the activation of JA signaling by NaCl and its effect on primary root growth. We found that JA-responsive JAZ genes were up-regulated by salt stress in a COI1-dependent manner in the roots. Using a JA-Ile sensor we demonstrated that activation of JA signaling by salt stress occurs in the meristematic zone and stele of the differentiation zone and that this activation was dependent on JAR1 and proteasome functions. Another finding is that the elongation zone (EZ) and its cortical cells were significantly longer in JA-related mutants (AOS, COI1, JAZ3 and MYC2/3/4 genes) compared with wild-type plants under salt stress, revealing the participation of the canonical JA signaling pathway. Noteworthy, osmotic stress - a component of salt stress - inhibited cell elongation in the EZ in a COI1-dependent manner. We propose that salt stress triggers activation of the JA signaling pathway followed by inhibition of cell elongation in the EZ. We have shown that salt-inhibited root growth partially involves the jasmonate signaling pathway in Arabidopsis.

Understanding protein arginine methyltransferase 1 (PRMT1) product specificity from molecular dynamics.

Protein arginine methyltransferases (PRMTs) catalyze the post-translational methylation of specific arginyl groups within targeted proteins to regulate fundamental biological responses in eukaryotic cells. The major Type I PRMT enzyme, PRMT1, strictly generates monomethyl arginine (MMA) and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). Multiple diseases can arise from the dysregulation of PRMT1, including heart disease and cancer, which underscores the need to elucidate the origin of product specificity. Molecular dynamics (MD) simulations were carried out for WT PRMT1 and its M48F, H293A, H293S, and H293S-M48F mutants bound with S-adenosylmethionine (AdoMet) and the arginine substrate in an unmethylated or methylated form. Experimental site-directed mutagenesis and analysis of the resultant products were also performed. Two specific PRMT1 active site residues, Met48 and His293, have been determined to play a key role in dictating product specificity, as: (1) the single mutation of Met48 to Phe enabled PRMT1 to generate MMA, ADMA, and a limited amount of SDMA; (2) the single mutation of His293 to Ser formed the expected MMA and ADMA products only; whereas (3) the double mutant H293S-M48F-PRMT1 produced SMDA as the major product with limited amounts of MMA and ADMA. Calculating the formation of near-attack conformers resembling SN2 transition states leading to either the ADMA or SDMA products finds that Met48 and His293 may enable WT PRMT1 to yield ADMA exclusively by precluding MMA from binding in an orientation more conducive to SDMA formation, i.e., the methyl group bound at the arginine Nη2 position.

A remodeled protein arginine methyltransferase 1 (PRMT1) generates symmetric dimethylarginine.

Protein arginine methylation is emerging as a significant post-translational modification involved in various cell processes and human diseases. As the major arginine methylation enzyme, protein arginine methyltransferase 1 (PRMT1) strictly generates monomethylarginine and asymmetric dimethylarginine (ADMA), but not symmetric dimethylarginine (SDMA). The two types of dimethylarginines can lead to distinct biological outputs, as highlighted in the PRMT-dependent epigenetic control of transcription. However, it remains unclear how PRMT1 product specificity is regulated. We discovered that a single amino acid mutation (Met-48 to Phe) in the PRMT1 active site enables PRMT1 to generate both ADMA and SDMA. Due to the limited amount of SDMA formed, we carried out quantum mechanical calculations to determine the free energies of activation of ADMA and SDMA synthesis. Our results indicate that the higher energy barrier of SDMA formation (ΔΔG(‡) = 3.2 kcal/mol as compared with ADMA) may explain the small amount of SDMA generated by M48F-PRMT1. Our study reveals unique energetic challenges for SDMA-forming methyltransferases and highlights the exquisite control of product formation by active site residues in the PRMTs.

Benchmarking Continuum Solvent Models for Keto-Enol Tautomerizations.

Experimental free energies of tautomerization, ΔGT, were used to benchmark the gas-phase predictions of 17 different quantum mechanical methods and eight basis sets for seven keto-enol tautomer pairs dominated by their enolic form. The G4 method and M06/6-31+G(d,p) yielded the most accurate results, with mean absolute errors (MAE's) of 0.95 and 0.71 kcal/mol, respectively. Using these two theory levels, the solution-phase ΔGT values for 23 unique tautomer pairs composed of aliphatic ketones, ß-dicarbonyls, and heterocycles were computed in multiple protic and aprotic solvents. The continuum solvation models, namely, polarizable continuum model (PCM), polarizable conductor calculation model (CPCM), and universal solvation model (SMD), gave relatively similar MAE's of ∼1.6-1.7 kcal/mol for G4 and ∼1.9-2.0 kcal/mol with M06/6-31+G(d,p). Partitioning the tautomer pairs into their respective molecular types, that is, aliphatic ketones, ß-dicarbonyls, and heterocycles, and separating out the aqueous versus nonaqueous results finds G4/PCM utilizing the UA0 cavity to be the overall most accurate combination. Free energies of activation, ΔG(‡), for the base-catalyzed keto-enol interconversion of 2-nitrocyclohexanone were also computed using six bases and five solvents. The M06/6-31+G(d,p) reproduced the ΔG(‡) with MAE's of 1.5 and 1.8 kcal/mol using CPCM and SMD, respectively, for all combinations of base and solvent. That specific enolization was previously proposed to proceed via a concerted mechanism in less polar solvents but shift to a stepwise mechanism in more polar solvents. However, the current calculations suggest that the stepwise mechanism operates in all solvents.

Exploring the catalytic mechanism of alkanesulfonate monooxygenase using molecular dynamics.

The complex mechanistic properties of alkanesulfonate monooxygenase (SsuD) provide a particular challenge for identifying catalytically relevant amino acids. In response, a joint computational and experimental study was conducted to further elucidate the SsuD mechanism. Extensive unbiased molecular dynamics (MD) simulations were performed for six SsuD systems: (1) substrate-free, (2) bound with FMNH2, (3) bound with a C4a-peroxyflavin intermediate (FMNOO(-)), (4) bound with octanesulfonate (OCS), (5) co-bound with FMNH2 and OCS, and (6) co-bound with FMNOO(-) and OCS. A previous theoretical study suggested that salt bridges between Arg297 and Glu20 or Asp111 initiated conformational changes critical for catalysis. However, our MD simulations and steady-state kinetic experiments did not corroborate this result. Similar kcat/Km values for both the E20A and D111A SsuD variants to wild-type SsuD suggest that the salt bridges are not critical to the desulfonation mechanism. Instead, the predicted role of Arg297 is to favorably interact with the phosphate group of the reduced flavin. Concomitantly, Arg226 functioned as a "protection" group shielding FMNOO(-) from bulk solvent and was more pronounced when both FMNOO(-) and OCS were bound. The stabilization of FMNOO(-) through electrostatic interactions with Arg226 would properly position the C4a peroxy group for the proposed nucleophilic attack on the sulfur of octanesulfonate.

Exploring the aldol reaction using catalytic antibodies and "on water" organocatalysts from QM/MM calculations.

The aldol reaction between benzaldehyde and acetone has been investigated using QM/MM Monte Carlo calculations and free-energy perturbation theory to determine the origin of the enhanced rates and enantioselectivities (% ee) derived from an enamine-based catalytic antibody 33F12 and a chiral organocatalyst. Electrostatic stabilization of the general acid/base TyrL36 by TrpH103, SerH100, and AsnL34 enabled the 33F12 active site to exclusively adopt an si-face benzaldehyde orientation for C-C bond formation with the LysH93-enamine. Whereas preorganization was responsible for the exclusive (S)-aldol product in the antibody, the organocatalyst featuring a chiral diphenyl amino alcohol moiety instead derived its preferred (R)-aldol product from an interplay between sterics and electronic stabilization. The si-face benzaldehyde conformation had unfavorable interactions with the organocatalyst in contrast to the re-face. Gas-phase calculations predicted a 73% ee; however, solution boosted the % ee values despite similar reaction geometries. An "on water" environment, defined as a reaction that proceeds in an aqueous organic emulsion, yielded a computed 94% ee (exptl 93% ee) compared to a calculated 87% ee in "neat" acetone (exptl 85% ee). Specific hydrogen bonding between the interfacial waters and an amide oxygen on the catalyst was found to control the % ee. A more compact si-face transition structure reduced solvent accessibility to the amide oxygen with a "closed state" steric barrier compared to an "open state" for the re-face. New insight into the synthetically important aldol reaction and state-of-the-art methodology is presented herein.

Simulating chemical reactions in ionic liquids using QM/MM methodology.

The use of ionic liquids as a reaction medium for chemical reactions has dramatically increased in recent years due in large part to the numerous reported advances in catalysis and organic synthesis. In some extreme cases, ionic liquids have been shown to induce mechanistic changes relative to conventional solvents. Despite the large interest in the solvents, a clear understanding of the molecular factors behind their chemical impact is largely unknown. This feature article reviews our efforts developing and applying mixed quantum and molecular mechanical (QM/MM) methodology to elucidate the microscopic details of how these solvents operate to enhance rates and alter mechanisms for industrially and academically important reactions, e.g., Diels-Alder, Kemp eliminations, nucleophilic aromatic substitutions, and ß-eliminations. Explicit solvent representation provided the medium dependence of the activation barriers and atomic-level characterization of the solute-solvent interactions responsible for the experimentally observed "ionic liquid effects". Technical advances are also discussed, including a linear-scaling pairwise electrostatic interaction alternative to Ewald sums, an efficient polynomial fitting method for modeling proton transfers, and the development of a custom ionic liquid OPLS-AA force field.

An ionic liquid dependent mechanism for base catalyzed ß-elimination reactions from QM/MM simulations.

Ionic liquids have been proposed to induce a mechanistic change in the reaction pathway for the fundamentally important base-induced ß-elimination class compared to conventional solvents. The role of the reaction medium in the elimination of 1,1,1-tribromo-2,2-bis(3,4-dimethoxyphenyl)ethane via two bases, piperidine and pyrrolidine, has been computationally investigated using methanol and the ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate and hexafluorophosphate [BMIM][BF(4)] and [BMIM][PF(6)], respectively. QM/MM Monte Carlo simulations utilizing free-energy perturbation theory found the ionic liquids did produce a reaction pathway change from an E1cB-like mechanism in methanol to a pure E2 route that is consistent with experimental observations. The origin of the ionic liquid effect has been found as: (1) a combination of favorable electrostatic interactions, for example, bromine-imidazolium ion, and (2) π-π interactions that enhance the coplanarity between aromatic rings maximizing the electronic effects exerted on the reaction route. Solute-solvent interaction energies have been analyzed and show that liquid clathrate solvation of the transition state is primarily responsible for the observed mechanistic changes. This work provides the first theoretical evidence of an ionic liquid dependent mechanism and elucidates the interplay between sterics and electrostatics crucial to understanding the effect of these unique solvents upon chemical reactions.

Design, development and evaluation of novel dual PPARδ/PPARγ agonists.

Type 2 diabetes is at epidemic proportions and thus development of novel pharmaceutical therapies for improving insulin sensitivity has become of paramount importance. The objectives of the current study were to develop novel dual PPARγ/δ agonists without the deleterious side effects associated with full PPARγ agonists. Docking simulations of 23 novel compounds within the ligand binding domain of PPARγ/δ were performed using AutoDock Vina which consistently reproduced experimental binding poses from known PPAR agonists. Comparisons were made and described with other docking programs AutoDock and Surflex-Dock (from SYBYL-X). Biological evaluation of compounds was accomplished by transcriptional promoter activity assays, quantitative PCR gene analysis for known PPARγ/δ targets as well as in vitro assays for lipid accumulation and mitochondrial biogenesis verses known PPAR agonists. We found one (compound 9) out of the 23 compounds evaluated, to be the most potent and selective dual PPARγ/δ agonist which did not display the deleterious side effects associated with full PPARγ agonists.

Uncovering the Critical Factors that Enable Extractive Desulfurization of Fuels in Ionic Liquids and Deep Eutectic Solvents from Simulations.

Environmental regulatory agencies have implemented stringent restrictions on the permissible levels of sulfur compounds in fuel to reduce harmful emissions and improve air quality. Problematically, traditional desulfurization methods have shown low effectiveness in the removal of refractory sulfur compounds, e.g., thiophene (TS), dibenzothiophene (DBT), and 4-methyldibenzothiophene (MDBT). In this work, molecular dynamics (MD) simulations and free energy perturbation (FEP) have been applied to investigate the use of ionic liquids (ILs) and deep eutectic solvents (DESs) as efficient TS/DBT/MDBT extractants. For the IL simulations, the selected cation was 1-butyl-3-methylimidazolium [BMIM] and the anions included chloride [Cl], thiocyanate [SCN], tetrafluoroborate [BF4], hexafluorophosphate [PF6], and bis(trifluoromethylsulfonyl)amide [NTf2]. The DESs were composed of choline chloride with ethylene glycol (CCEtg) or with glycerol (CCGly). Calculation of excess chemical potentials predicted the ILs to be more promising extractants with energies lower by 1-3 kcal/mol compared to DESs. Increasing IL anion size was positively correlated to enhanced solvation of S-compounds, which was influenced by energetically dominant solute-anion interactions and favorable solute-[BMIM] π-π stacking. For the DESs, the solvent components offered a range of synergistic, yet comparatively weaker, electrostatic interactions that included hydrogen bonding and cation-π interactions. An in-depth analysis of the structure of IL and DES systems is presented, along with a discussion of the critical factors behind experimental trends of S-compound extraction efficiency.

The Carrot Phytoene Synthase 2 (DcPSY2) Promotes Salt Stress Tolerance through a Positive Regulation of Abscisic Acid and Abiotic-Related Genes in Nicotiana tabacum.

Background: Carotenoids, which are secondary metabolites derived from isoprenoids, play a crucial role in photo-protection and photosynthesis, and act as precursors for abscisic acid, a hormone that plays a significant role in plant abiotic stress responses. The biosynthesis of carotenoids in higher plants initiates with the production of phytoene from two geranylgeranyl pyrophosphate molecules. Phytoene synthase (PSY), an essential catalytic enzyme in the process, regulates this crucial step in the pathway. In Daucus carota L. (carrot), two PSY genes (DcPSY1 and DcPSY2) have been identified but only DcPSY2 expression is induced by ABA. Here we show that the ectopic expression of DcPSY2 in Nicotiana tabacum L. (tobacco) produces in L3 and L6 a significant increase in total carotenoids and chlorophyll a, and a significant increment in phytoene in the T1L6 line. Tobacco transgenic T1L3 and T1L6 lines subjected to chronic NaCl stress showed an increase of between 2 and 3- and 6-fold in survival rate relative to control lines, which correlates directly with an increase in the expression of endogenous carotenogenic and abiotic-related genes, and with ABA levels. Conclusions: These results provide evidence of the functionality of DcPSY2 in conferring salt stress tolerance in transgenic tobacco T1L3 and T1L6 lines.

Free Energy Calculations and Conformational Analysis of Dibenzo-30-crown-10 with Sm2+, Eu2+, and Three Halide Salts in THF Using the AMOEBA Force Field.

Crown ether complexes have been tailored for use in industrial separations of lanthanides (Ln) as a part of rare earth mining and refining. Dibenzo-30-crown-10 (DB30C10) is one of the most efficient complexants for the separation of rare earth mixtures based on the cation size. To understand the origin of this complexation, molecular dynamics (MD) simulations of DB30C10 have been performed using different combinations of divalent Sm and Eu and three halide salts Cl-, Br-, and I- in tetrahydrofuran (THF) solvent. DB30C10 was parameterized here for the polarizable atomic multipole optimized energetics for biomolecular simulation (AMOEBA) force field, and the existing parameters of THF, Sm2+, and Eu2+ were employed from our previous efforts. The large conformational fluctuations present in the DB30C10 systems were found to be dependent both on the identity of the lanthanide and halide complexes. For Cl- and Br- systems, there were no observed conformational changes at 200 ns, while in I- systems, there were two conformational changes with Sm2+ and one with Eu2+ within that same timeframe. In SmI2-DB30C10, there were three stages of conformational changes. In the first stage, the molecule is unfolded, in the second stage, the molecule is partly folded, and finally, in the third stage, the molecule is completely folded. Lastly, the Gibbs binding free energies of DB30C10 with SmBr2 and EuBr2 have been computed, which resulted in nearly identical ΔGcomp values for each lanthanide with Sm2+ being slightly more favorable. Considering the folding mechanism of the SmI2 system with DB30C10, the Gibbs binding free energies of DB30C10 and dicyclohexano-18-crown-6 (DCH18C6) with SmI2 were calculated separately and compared to probe their complexation affinities, in which the former was found to be more favorable.

ABA Biosynthesis- and Signaling-Related Gene Expression Differences between Sweet Cherry Fruits Suggest Attenuation of ABA Pathway in Bicolored Cultivars.

Fruit development involves exocarp color evolution. However, signals that control this process are still elusive. Differences between dark-red and bicolored sweet cherry cultivars rely on MYB factor gene mutations. Color evolution in bicolored fruits only occurs on the face receiving sunlight, suggesting the perception or response to color-inducing signals is affected. These color differences may be related to synthesis, perception or response to abscisic acid (ABA), a phytohormone responsible for non-climacteric fruit coloring. This work aimed to determine the involvement of ABA in the coloring process of color-contrasting varieties. Several phenolic accumulation patterns differed between bicolored 'Royal Rainier' and dark-red 'Lapins'. Transcript abundance of ABA biosynthetic genes (PavPSY, PavZEP and PavNCED1) decreased dramatically from the Pink to Red stage in 'Royal Rainier' but increased in 'Lapins', which correlated with a higher ABA content in this dark-red cultivar. Transcripts coding for ABA signaling (PavPP2Cs, PavSnRKs and PavMYB44.1) were almost undetectable at the Red stage in 'Royal Rainier'. Field trials revealed that 'Royal Rainier' color development was insensitive to exogenous ABA, whereas it increased in 'Lapins'. Furthermore, ABA treatment only increased transcript levels of signaling genes in 'Lapins'. Further studies may address if the ABA pathway is attenuated in bicolor cultivars.